Plant Genome Manipulation

Question 1

What are transgenic plants, and when were they first created?

Answer 1

Transgenic plants contain genes introduced from an unrelated source. They were first developed in 1983 using Agrobacterium tumefaciens in tobacco, sunflower, and petunia.

Question 2

What is transformation in plants, and how is it achieved?

Answer 2

Transformation introduces foreign genes into plants via non-sexual methods. This involves: 1. Incorporation of DNA into the genome. 2. Use of selectable markers (e.g., antibiotic resistance) to identify transformed cells.

Question 3

What is Agrobacterium tumefaciens, and why is it important in genetic engineering?

Answer 3

Agrobacterium tumefaciens is a soil bacterium that naturally causes crown gall disease. Its Ti plasmid can be modified to deliver foreign genes into plant cells.

Question 4

What are the key components of the Ti plasmid used in plant transformation?

Answer 4

1. T-DNA: Transferred DNA flanked by left and right borders. 2. Vir Genes: Mediate T-DNA transfer into plant cells. 3. Oncogenes and opine synthesis genes (replaced during modification).

Question 5

How was Agrobacterium’s role in crown gall formation discovered?

Answer 5

Bacteria isolated from galls could induce tumors in other plants, proving the bacterial transfer of a tumor-inducing principle (T-DNA).

Question 6

What are opines, and why are they significant?

Answer 6

Opines are metabolites produced in tumor tissues due to T-DNA transfer. They serve as a nutrient source for Agrobacterium but are not metabolized by plants.

Question 7

What is a binary vector system in Agrobacterium-mediated transformation?

Answer 7

The Ti plasmid is split into: 1. T-DNA vector: Contains the gene of interest and selectable marker. 2. Helper plasmid: Provides virulence (Vir) genes for T-DNA transfer.

Question 8

What are chimeric genes, and how are they constructed?

Answer 8

Chimeric genes combine a promoter and gene of interest. Example: CaMV 35S promoter (from cauliflower mosaic virus) drives high-level expression.

Question 9

Why are selectable markers used in plant transformation?

Answer 9

Selectable markers (e.g., antibiotic resistance genes like kanamycin) ensure that only successfully transformed cells survive and regenerate.

Question 10

What is the forward genetics approach?

Answer 10

Forward genetics identifies genes responsible for a phenotype: 1. Mutagenesis → screen mutants. 2. Test heritability. 3. Map and identify the causal gene.

Question 11

What is the reverse genetics approach?

Answer 11

Reverse genetics starts with a known gene to determine its function by: 1. Knockout (inactivation). 2. Overexpression. 3. Studying mutant phenotypes.

Question 12

How does T-DNA insertion create mutants in plants?

Answer 12

T-DNA randomly inserts into the genome, often disrupting gene function. This allows researchers to identify gene roles based on resulting mutant phenotypes.

Question 13

How can promoter swapping help identify gene function?

Answer 13

By replacing a gene’s promoter, researchers can control where and when the gene is expressed, revealing its role.

Question 14

What are reporter genes, and how are they used?

Answer 14

Reporter genes (e.g., GUS, GFP, luciferase) produce detectable signals to: - Identify gene activity in specific cells. - Visualize protein localization.

Question 15

Why is GFP a popular reporter gene in plant studies?

Answer 15

GFP fluoresces under light, allowing live, cell-specific imaging without requiring substrates. It is small and does not disrupt protein function.

Question 16

What was revealed through experiments on SEP genes?

Answer 16

Triple mutants in SEPALLATA 1-3 demonstrated their role in floral organ identity. Overexpression (35S::SEP3) revealed their redundancy in flower development.

Question 17

How is Rhizobium modified for transformation, and why?

Answer 17

Rhizobium can be engineered to carry Ti plasmids to avoid Agrobacterium-related patent issues, providing an alternative transformation vector.

Question 18

Describe the steps in T-DNA transfer to plant cells.

Answer 18

1. Plant wounds release signals → activate VirA/VirG genes. 2. T-DNA is cut and transferred through the Type IV secretion system. 3. T-DNA integrates into the plant genome.

Question 19

What is the RUBY reporter system, and why is it useful?

Answer 19

RUBY produces red pigment in transformed plants, providing a non-invasive visual marker to identify successful transformation.

Question 20

What are the challenges in manipulating plant genomes?

Answer 20

1. Difficulty in targeted gene editing (e.g., homologous recombination). 2. Tissue-specific gene regulation. 3. Public concerns about GM crops.

Question 21

What experiment confirmed T-DNA transfer from Agrobacterium?

Answer 21

Restriction enzyme digestion showed T-DNA integrates into plant DNA, confirming bacterial transformation of host cells.

Question 22

How was the Ti plasmid engineered for plant transformation?

Answer 22

Oncogenes and opine synthesis genes were replaced with selectable markers and genes of interest.

Question 23

Briefly describe three different reporter genes commonly used in plants, including their species of origin and how their activity is monitored in transgenic plants. For each reporter, explain one advantage and one disadvantage to its use.

Answer 23

1. GUS (β-glucuronidase)
   
   • Species of origin: Escherichia coli (bacterium)
   • Monitoring: GUS activity is monitored by staining the plant tissues with a substrate like X-gluc, which is cleaved by GUS to produce a blue or colorless product, allowing visualization of gene expression.
   • Advantage: Simple and inexpensive to use; can provide clear and visible results.
   • Disadvantage: Requires the use of chemical substrates that may not be ideal for all tissues or developmental stages, and its use may be toxic in some plant species.
2. GFP (Green Fluorescent Protein)
   
   • Species of origin: Aequorea victoria (jellyfish)
   • Monitoring: GFP fluoresces green when exposed to ultraviolet (UV) or blue light, allowing real-time, non-invasive monitoring of gene expression in living tissues.
   • Advantage: Enables in vivo visualization of gene expression in real time without the need for substrates or staining.
   • Disadvantage: Fluorescence can be weak in some tissues, and the technique requires specialized equipment such as fluorescence microscopes.
3. Luciferase
   
   • Species of origin: Photinus pyralis (firefly)
   • Monitoring: Luciferase catalyzes the oxidation of luciferin, producing light, which can be detected using a luminometer to monitor gene expression.
   • Advantage: Provides sensitive and quantitative measurement of gene expression.
   • Disadvantage: Requires the addition of luciferin as a substrate, and its use can be limited to assays where living tissues can be collected or analyzed.